Proc. NatL Acad. Sci. USAVol. 80, pp. 1290-1294, March 1983Botany

An Arabidopsis thaliana mutant defective in chloroplastdicarboxylate transport

(photorespiration/malate shuttle/glutamine transport)

S. C. SOMERVILLE*t AND W. L. OGREN*t*Department of Agronomy, University of Illinois, Urbana, Illinois 61801; and tU. S. Department of Agriculture, Agricultural Research Service, Urbana, Illinois 61801

Communicated by Harry Beevers, November 8, 1982

ABSTRACT Reactions of the photorespiratory pathway of C3plants are found in three subcellular organelles. Transport pro-cesses are, therefore, particularly important for maintaining theuninterrupted flow of carbon through this pathway. We describehere the isolation and characterization of a photorespiratory mu-tant of Arabidopsis thaliana defective in chloroplast dicarboxylatetransport. Genetic analysis indicates the defect is due to a simple,recessive, nuclear mutation. Glutamine and inorganic phosphatetransport are unaffected by the mutation. Thus, in contrast to pre-vious reports for pea and spinach, glutamine uptake by Arabi-dopsis chloroplasts is mediated by a transporter distinct from thedicarboxylate transporter. Both the inviability and the disruptionof amino-group metabolism of the mutant under photorespiratoryconditions suggest that the primary function of the dicarboxylatetransporter in vivo is the transfer of 2-oxoglutarate and glutamateacross the chloroplast envelope in conjunction with photorespira-tory nitrogen metabolism. The role commonly ascribed to thistransporter, conducting malate-aspartate exchanges for the in-direct export of reducing equivalents from the chloroplast, ap-pears to be a minor one.

The constituent reactions of the photorespiratory pathway ofhigher plants occur in three organelles, the chloroplast, the mi-tochondrion, and the peroxisome. Thus, transport processes mustintervene at several steps of the pathway (Fig. 1) (1, 2). To theextent that the transport of substrates, products, or cofactors islimiting, these transport processes may be expected to exert astrong regulatory influence on photorespiratory metabolism.The photorespiratory pathway is initiated by the oxygenation

of ribulose bisphosphate by the bifunctional enzyme ribulose-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) (3, 4). 02and CO2 act as competitive substrates for this enzyme, and thedegree to which carbon is diverted from the Calvin cycle to thephotorespiratory pathway is a function of the ratio of these twogases in the atmosphere (5). Experimentally, the flux of carbonthrough the pathway can be suppressed without adverse effectby placing plants in an atmosphere enriched in CO2 or enhancedby transferring plants to an atmosphere enriched in 02(6). Three-quarters of the carbon entering the pathway is returned to thepool of Calvin-cycle intermediates as phosphoglycerate. The re-maining carbon is lost as CO2 at the glycine decarboxylase stepin the mitochondrion.

Tightly integrated with the photorespiratory carbon cycle isthe photorespiratory nitrogen cycle in which ammonia releasedduring glycine deamination is refixed by the sequential actionofglutamine synthetase and glutamate synthase (Fig. 1) (7). Theresultant glutamate supports both photorespiratory ammoniarefixation (8) and glyoxylate amination (9). In the absence ofadequate glutamate pools, glyoxylate is oxidized nonenzymat-

ically to CO2 at rates that significantly reduce net CO2 assim-ilation (9), and ammonia accumulates to toxic levels (8). Becauseglutamate is synthesized in the chloroplast (7) and consumedin the peroxisome, the transfer ofglutamate and 2-oxoglutaratebetween these two organelles is a necessary component ofpho-torespiratory nitrogen metabolism.

Transport systems operating at the peroxisome-boundingmembrane have not been characterized. However, studies withisolated chloroplasts have revealed the presence of a trans-porter, designated the dicarboxylate transporter, which cata-lyzes the counter-exchange of several dicarboxylic acids acrossthe chloroplast inner membrane (10). These compounds includemalate, 2-oxoglutarate, aspartate, and glutamate (10). Gluta-mine is also a reported substrate for this carrier (11, 12). There-fore, the chloroplast dicarboxylate transporter is implicated asan important component of the photorespiratory pathway. Be-cause this transporter is also capable of effecting malate-as-partate exchanges, it has been ascribed the role of mediating theindirect export of reducing equivalents to the cytoplasm (13,14).The isolation and characterization of a photorespiratory mu-

tant defective in chloroplast dicarboxylate transport is reportedhere. Biochemical and physiological analyses of the mutant haveproven useful in determining the specificity of this transporterand in ascertaining its primary in vivo role.

MATERIALS AND METHODSPlant Material and Culture. Both the mutant line CS156, the

subject of this study, and the previously described line CS113,a glutamate synthase-deficient mutant (8), were recovered in ascreen for mutants of Arabidopsis thaliana (L.) Heynh. (raceColumbia) with defects in photorespiratory metabolism (6). Thebasis of the mutant selection procedure is that strains with de-fects in the photorespiratory pathway cannot survive at atmo-spheric levels of CO2 and 02 but grow normally at 1% CO2, whenphotorespiration is suppressed.

Plants were grown according to described methods and con-ditions (15). For most of this study, a line descended from abackcross of CS156 to the wild type was used. Experiments wereconducted with plants at the rosette stage of development (3-4 wk from seeding). Procedures for making genetic crosses andmeasuring gas exchange have been described (15).

Labeling Studies. Plants were labeled with 14CO2 for 10 min,and the distribution of label among products was determinedby ion-exchange and thin-layer chromatography (15-17).

Glutamate Synthase Assays. Glutamate synthase was as-sayed in crude extracts of leaf material after centrifugation at

Abbreviation: Chl, chlorophyll.tPresent address: MSU-DOE Plant Research Laboratory, Michigan StateUniv., East Lansing, MI 48824.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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KrebsCycy

APGADP

L3-dPGA

TP

Sucros

Mdote

OAA

-2064

Asp

TPCH

I,-diPGA

PGA C02

P-Glycolate

I___________________ I__________

HLOROPLAST

amg,hb

,NWCOHNH3

FIG. 1. Schematic presentation of thephotorespiratory carbon andnitrogen cycles. C1-THF, N5,N'0-methylenetetrahydrofolate; DT, di-carboxylate transporter; OAA, oxaloacetate; 20G, 2-oxoglutarate; PT,phosphate translocator; PGA, phosphoglycerate; RuBP, ribulose bis-phosphate; THF, tetrahydrofolate; TP, triose phosphate.

30,000 x g and desalting by Sephadex G-25 column chroma-tography (8). Protein was determined by a dye-binding assay

(18) with bovine serum albumin as the standard.Ammonia and Amino Acid Determinations. Plants were

equilibrated. in darkness for 25 min and then illuminated (300microeinsteins m-2se&- of photosynthetically active radiation)for various lengths of time in an open gas exchange system main-tained at 25TC and 70% relative humidity. The photorespiratorygas regime was nitrogen containing 357 1.l of CO2 liter' and49% 02. Methods for quantitating ammonia and amino acid lev-els in leaf tissue have been described (8). Amino acid values werenormalized by using the amount of phenylalanine per mg ofchlorophyll (Chl) as a basis to correct for losses that occurredduring the preparation of some samples.

Chloroplast Isolation and Transport Assays. Chloroplasts wereprepared from Percoll gradient-purified protoplasts (19) and usedimmediately. The intactness of the chloroplast preparations wasdetermined by the ferricyanide test (20).

Oxoglutarate + glutamine-dependent 02 evolution by iso-lated chioroplasts was measured in a Hansatech 02 electrode at25°C at a light intensity of 1,000 microeinsteins m 2sec- ofphotosynthetically active radiation at the surface of the cuvette(21). Chloroplasts (20-40 ,ug of Chl) were added to the standardassay medium [300 mM sorbitol/28 mM HepesrKOH, pH 7.6/10 mM NaHCO3/2.5 mM EDTA/0.15 mM potassium phos-phate/0. 1%. fatty acid-free bovine serum albumin/300 units ofcatalase per ml (19)]. D,L-Glyceraldehyde was added to 6-10

m.M to inhibit C02-dependent 02 evolution (22).The silicone oil layer filter centrifugation technique was used

to measure the transport of radiolabeled compounds into freshlyisolated, intact chloroplasts (23). The standard assay mediumcontained, in addition to the standard components, 10-30 tigof Chl and 1-3 ,uCi (3.7-11.1 x 104 Bq) of 3H20. Glutaminetransport was determined at pH 7.9. The silicone oil layer con-sisted of AR200/AR20 60:40 (wt/wt) (Wacker Chemie, SWSSilicones, Adrian, MI). Chloroplast volumes were determinedwith [14C]sorhitol in parallel experiments under the same con-ditions as the. transport assays (23). The average sorbitol-im-permeable space was 38 ,ul/mg of Chl and 44 p.l/mg of Chl forwild-type and mutant chloroplasts, respectively: Transport as-says, commonly 3-, 5-, or 10-sec duration, were performed inthe dark at 50C. For some experiments, chloroplasts were pre-loaded with the compound to be assayed for uptake by addinga small volume of stock solution to freshly prepared chloroplaststo give a final concentration of 20 mM. After 15 min on ice inthe dark, the chloroplasts were collected by centrifugation (270X g at 40C for 40 sec) and resuspended in medium lacking thecompound. Apparent Km and Vm.x values were estimated fromScatchard plots of the data presented in the text. Chl was de-termined spectrophotometrically in ethanol (24).

RESULTSMutant Isolation and Genetic Analysis. In an atmosphere that

suppressed photorespiration (1% C02/99% air), the mutant lineCS156 was capable of normal growth and development. How-ever, in standard atmospheric conditions (N2 containing 0.03%CO2 and 21% 02), the mutant became yellow and lost vigor within3-4 days. The F1 plants from a CS156 x wild-type cross werehealthy in standard atmospheres, suggesting the mutant linecarried a recessive, nuclear mutation. In a derivative F2 pop-ulation, 196 plants exhibited the wild-type phenotype and 55were yellow after 4 days in a normal atmosphere. Thus, the mu-tation in line CS 156 responsible for the growth requirement forhigh CO2 was inherited as a simple, recessive, nuclear mutation(X2 = 1.276; P> 0.25). The locus defined by this mutant wasdesignated dct (dicarboxylate transport). The F1 plants from across of CS 156 with CS 113, a glutamate synthase-deficient line(8), were normal in appearance and capable of sustained growthin a normal gas regime. Because the mutation in CS156 com-plemented that in CS 113, the dct locus was genetically distin-guished from the gluS locus.

Gas Exchange Analyses. CO2 fixation was measured on in-dividual plants in nonphotorespiratory (Fig. 2A) and photores-piratory (Fig. 2B) gas regimes. The photosynthesis rate of themutant exceeded that of the wild type in an atmosphere of low02 concentration (Fig. 2A). In contrast, photosynthesis was im-paired in the mutant in atmospheres that promoted photores-piration. In the photorespiratory gas regime of nitrogen con-taining.49% 02 and 357 A.l of C02 liter', the final rate of CO2-fixation in CS156.was reduced to 27% of the rate in wild type(Fig. 2B). CO2 evolution into C02-free 50% 02/50% N2, a mea-sure of photorespiration, also was reduced in mutant plants (datanot presented). Thus, the lesion in mutant CS156 disruptedphotorespiratory metabolism specifically and had no detectableeffect on photosynthesis and growth in nonphotorespiratory en-vironments.

Metabolite Distribution. A defect in photorespiratory me-tabolism was evident in the mutant from the altered distributionpattern of metabolites labeled with '4Co2 for 10 min in a pho-torespiratory gas regime (Table 1). Notably, the accumulationof 14C label in the basic fraction was reduced and that found inthe acid-i fraction was increased in mutant plants compared to

ItMabte Glycerote GlyWcote

OAA OHPyruvate Qlyoxyote

'4 >342:

-Asp Sor Gly

PEROXISOE T I

I x^ASer * Gly

THF T

MITOCHONDRIONNAM NOD

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20

10

cq

0

C,

; 20

0bo

= 10

¢I

0~

0

B

0 20 40 60 80Time, min

FIG. 2. NetCO2fixationbywild!typeandmutantArabidopsisplantsunder nonphotorespiratory (A) and photorespiratory (B) conditions. Thegas regime inA was nitrogen containing 352 gl of C02 liter-' and 2%02 and inB was nitrogen containing 357 AI of C02 liter-' and 49% 02.-* Response of wild type; ---, response of mutant CS156;-; dark

conditions; m, light conditions. The response shown represents the av-erage of two experiments.

wild-type plants. Labeling of all major constituents of the basicfraction was depressed. The increase of 14C label in the acid-ifraction was primarily due to an increased labeling of 2-oxoglu-tarate. In these respects the labeling pattern of the dct mutantclosely resembled that of a glutamate synthase-deficient line,CS113 (Table 1), implicating a defect in glutamate synthesis as

the cause for the high CO2 requirement of the dct mutant.

Table 1. Percentage distribution of the products of14c0 assimilation*

Distribution (%) by strain

Fraction

BasicGlutamateGlycineSerine

Acid-iMalateGlycerate2-Oxoglutarate

Acid-2Acid-3NeutralInsoluble

Recovery

Wild type CS15641.01.5

21.25.86.33.61.0,0.

24.08.9

14.04.6

100.8

17.20.79.03.6

29.82.40

14.027.311.311.05.1

CS11316.30.5

10.02.2

22.62.60

8.733.513.110.86.2

101.7 102.5* Values given are the percentage of total 14C incorporated into leaves.Plants were labeled with 14C02 at 5-15 min from the onset of, illu-mination. The photorespiratory gas regime was as described, andtheconditions during labelingwere 25TC, 70% relative humidity, and 300microeinsteins m 2sec' of photosynthetically active radiation. Eachvalue is the mean of three determinations.

In consideration of the difficulties associated with equatingthe distribution of radioactive label. and'mass flow of metabo-lites, we undertook a quantitative analysis of amino acid poolsin the leaf under photorespiratory conditions. The results of thisexperiment confirmed that the mutant harbored a defect inglutamate metabolism (Fig. 3)'. Upon illumination, glutamatelevels in the mutant declined, suggesting that utilization of thisamino acid. greatly exceeded its synthesis (Fig. 3A)'. Glutaminelevels in the mutant declined slightly and then stabilized at arelatively high level (Fig. 3B). Lack of depletion of glutaminepools and accumulation of '4C label in 2-oxoglutarate (Table 1)implied that the mutant was not able to convert these two com-pounds to glutamate. Ammonia accumulated to abnormally highlevels in the mutant plants (Fig. 3C). A similar result was ob-served in the gluS. mutant, CS113, which. was unable to refixphotorespiratory ammonia by the glutamine synthetase/glu-tamate synthase reactions (8). The-metabolism of glycine (Fig.3D) and that of serine (Fig. 3E), both intermediates of the pho-torespiratory pathway, were also abnormal in the mutant line.These are considered secondary effects resulting from the dis-ruption of photorespiratory metabolism.

8.0 0 A

4.0

000 -

8.0 -o B.

4.0

00o

a0.2 0 0

0

4a)Q 0/-

D A~~D2 2.0.

1.0

0 j

E0

1.0

0 10 20 30'Time, min.

FIG. 3. Amino acid and ammonia levels in leaves of wild-type (o)and mutant CS156 (o) Arabidopsis plants under photorespiratory con-ditions. (A) Glutamate. (B) Glutamine. (C) Ammonia. (D) Glycine. (E)Serine.

Proc. Natl. Acad. Sci.-, USA 80 (1983)

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Proc. Natl. Acad. Sci. USA 80 (1983) 1293

Enzyme Analyses. Crude leaf extracts of mutant plants showed63% of wild-type levels of ferredoxin-dependent glutamate syn-thase activity (data not presented). However, previous analysesof gluS mutants showed that F1 (wild-type x gluS) plants with50% of wild-type levels of glutamate synthase activity werephenotypically normal (8). Thus, the observed reduction of en-

zyme activity in CS 156 was not sufficient to account for the highCO2 growth requirement in the mutant. Also, as noted above,the mutation in CS156 was not allelic to the gluS mutation inCS113.

Transport Assays. Physiological studies of metabolite poolsstrongly indicated glutamate metabolism was disrupted in thedct mutant, but in vitro enzyme assays did not verify this con-clusion. To examine this inconsistency, glutamate synthase wasmeasured in isolated, intact chloroplasts as 2-oxoglutarate +glutamine-dependent 02 evolution (21). At low external con-centrations of 2-oxoglutarate, no 2-oxoglutarate + glutamine-dependent 02 evolution by mutant chloroplasts was detected(Table 2). However, when the 2-oxoglutarate concentration wasraised to 100 mM, chloroplasts from both the mutant and wildtype exhibited glutamate synthase activity. Thus, it appearedthat the enzyme was present and active in chloroplasts from thedct mutant, confirming conclusions from in vitro enzyme as-says. On the basis of these experiments, it was considered prob-able that chloroplasts from the mutant were substantially lesspermeable to 2-oxoglutarate than were wild-type chloroplasts.The uptake of 2-oxoglutarate and several other compounds

by isolated chloroplasts was measured directly by using the sil-icone oil filter centrifugation technique (23). For wild-typechloroplasts, the uptake of four dicarboxylates, glutamine, andinorganic phosphate became saturated at high external sub-strate concentrations, a characteristic of facilitated transport (Fig.4). To better characterize the transporters in wild-type Arabi-dopsis chloroplasts, kinetic parameters describing the concen-

tration dependence of transport rates for these compounds weredetermined (Table 3). Apparent Km values were slightly higherthan those reported for spinach (10, 25) and pea (12). Values ofVma, for the four dicarboxylates and glutamine were consider-ably higher (10, 12). For chloroplasts from the mutant, thetransport of malate, 2-oxoglutarate, aspartate, and glutamatewas severely reduced. However, the uptake of glutamine wasindistinguishable from the wild type, suggesting that this aminoacid was transported by a carrier distinct from the dicarboxylatetransporter (Fig. 4E). The-uptake of inorganic phosphate wasunaffected by the lesion at the dct locus in the mutant (Fig. 4F).Similar results were obtained at 250C. Thus, it is unlikely thatthe loss of dicarboxylate transport activity was an artifact of low-temperature effects on the mutant chloroplast envelope. Also,the normal functioning of two carriers in the envelope of themutant indicated that the lesion was specific for dicarboxylatetransport and did notaffect thegeneral architecture of the chlo-.roplast envelope.

The chloroplast dicarboxylate transporter is reported to op-erate by a counterexchange mechanism (10). For some exper-

Table 2. Glutamine + 2-oxoglutarate-dependent 02 evolution byisolated chloroplasts

02 evolution, Amol/mgof Chl per hr

Substrate, mM Wild type CS156Glutamine 2-Oxoglutarate strain mutant

5 3 4.77 05 100 4.00 10.60

The percentage chloroplast intactness was 88% for wild typeand 76%for CS156.

120

0H_.4

S-4

-a)

80

40

0

0

A /-

0-

1.0 2.0

0

B

D 1.0 2.0

E

!8

0 1.0 2.0 0 4.0 8.0 0 0.4 0.8 1.2External concentration, mM

FIG. 4. The uptake of several compounds into the sorbitol-im-permeable space of chloroplasts from wild-type (e) and mutant CS156(0) plants. All experiments were conducted at 5YC. Chloroplasts usedin these experiments were >94% intact. The compounds assayed foruptake were 2-oxoglutarate (A), aspartate (B), glutamate (C), malate(D), glutamine (E), and inorganic phosphate (F). For experiments pre-sented in A, B, C, and D, chloroplasts were preloaded with the com-pound to be assayed for transport.

iments, chloroplasts were incubated in high concentrations ofsubstrate prior to measuring uptake to ensure that the internalsupply of dicarboxylate did not limit the uptake of exogenouslysupplied compounds by the counterexchange transporter. Pre-loading wild-type chloroplasts with malate, 2-oxoglutarate, andglutamate stimulated malate, 2-oxoglutarate, and glutamate up-take, respectively, =2-fold. This enhancement of transport ratecannot be attributed to significant carryover of substrate fromthe preloading to the assay step. The amount of carryover wascalculated to be 31-90 nmol (equivalent to a 0.010 to 0.028 mMincrease in the substrate concentration in the assay mixture) bycomparing the rate of glutamate or aspartate uptake at low ex-ternal concentrations not stimulated by preloading with the rateexpected when a linear relationship between transport rate andconcentration is assumed. In addition to enhancing the rate ofuptake and, hence, the observed Vmax, preloading led to a re-

duction by 0.5 mM of the apparent Km of 2-oxoglutarate andmalate for wild-type chloroplasts. Uptake of 2-oxoglutarate andglutamate by mutant chloroplasts was unaffected by the pre-

loading step. However, in the absence of preloading, malateuptake by the mutant could not be detected. A low rate of as-

partate uptake, which did not saturate at 2.0 mM, was observedwhen the preloading step was omitted. With preloading, a re-

duced level of apparently facilitated aspartate transport was

measured (Fig. 4B).

Table 3. Kinetic constants of uptake of several compounds bywild-type chloroplasts

VmLax, PMo0/mgSubstrate Km x 103 of Chl per hr

Malate 1.3 1302-Oxoglutarate 0.5 176Aspartate 0.5 111

Glutamate 2.7 150Glutamine 2.4 42Inorganic phosphate 0.5 90

C

/00T. fI

0 1.0 2.0 3.0

F

0~o'e

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DISCUSSIONMutant line CS156 carries a recessive nuclear mutation re-sponsible for the specific loss of chloroplast dicarboxylate trans-port. activity. Thus, although.the dct mutant showed substantialin vitro glutamate synthase activity, in vivo analyses indicatedthat the enzyme was essentially inoperative because of sub-strate limitation (Table 2 and Fig. 3).The lethality of the dct mutant in atmospheres promoting

photorespiration can be explained by the in vivo loss of gluta-mate synthase function. This in turn restricts glyoxylate ami-nation and photorespiratory ammonia refixation (Fig. 3C) bylimiting the supply of glutamate (Fig. 3A). If glyoxylate is nottransaminated, it does not accumulate but undergoes nonen'zymatic oxidation, resulting in, a greatly enhanced loss of re-cently fixed carbon. As a result, Calvin-cycle intermediates aredepleted, leading to a decline in net CO2 fixation (Fig. 2B). Sim-ilar explanations have been offered for the high CO2 growth re-quirement of four other classes of mutants with defects in pho-torespiratory nitrogen metabolism (8, 9, 26, 27).- These resultsemphasize the interdependence of the photorespiratory carbonand nitrogen cycles.

The chloroplast dicarboxylate transporter is generally con-sidered an essential component of a malate shuttle for trans-porting reducing equivalents across the chloroplast inner mem-brane (13, 14). Because mutant CS 156 was recovered in a screenfor photorespiratory mutants and was capable of normal pho-tosynthesis (Fig. 2A) and growth in nonphotorespiratory re-gimes, the major role of the chloroplast dicarboxylate trans-porter in vivo must be as a component of the photorespiratorynitrogen cycle. Conversely, the proposed role for this trans-porter as a part of the chloroplast malate shuttle appears to beof minor physiological importance. The chloroplast phosphatetranslocator is the only other known route for indirectly ex-porting reducing equivalents from the chloroplast at significantrates (14, 25). However, both ATP and NAD(P)H are exportedin an obligate manner by this transporter. It seems likely thatsome mechanism must exist for modulating the cytosolic levelsof ATP and NADH. The low level of dicarboxylate transportactivity observed in the dct mutant may be adequate to meetthis requirement.The dct mutant has been useful for determining the speci-

ficity of the dicarboxylate transporter. Previous studies basedon back-exchange (11) and competition (12) experiments sug-gested glutamine was transported across the chloroplast enve-lope by the dicarboxylate transporter. This appears to be in-consistent with the observation that glutamine transport isunaffected by the lesion at the dct locus in the mutant. Clearlya separate transporter for glutamine, distinct from the dicar-boxylate carrier, must occur in the chloroplast envelope. Thediscrepancy between previous and present results regarding thespecificity of the dicarboxylate carrier can be resolved if twocarriers with overlapping specificity exist in the chloroplast en.velope. The carrier defined by the dct mutant recognizes di-carboxylates but not glutamine (Fig. 4), whereas the second car-rier presumably transports dicarboxylates at a low rate andglutamine. The low level of malate, 2-oxoglutarate, glutamate,

and aspartate uptake by chloroplasts of the dctmutant may rep-resent transport activity by this second carrier. Aspartate (10)and 2-oxoglutarate (M. 0. Proudlove and D. A. Thurman, per-sonal communication) are recognized by more than one chlo-roplast carrier. In each case, the second transporter was de-scribed as having a relatively low activity.

Mutants harboring defective transporters generally have beenidentified as being resistant to an exogenously supplied toxicsubstance. For this reason, transport processes operating acrossthe plasma membrane of simple organisms have been the mostamenable to analysis with. mutants. The dot mutant of Arabi-dopsis demonstrates that intracellular transport in eukaryotesalso can be subject- to mutant analysis.

1. Chollet, R. & Ogren, W. L. (1975) Bot. Rev. 41, 137-179.2. Tolbert, N. E. (1979) in Encyclopedia of Plant Physiology, New

Series, eds. Gibbs, M. & Latzko, E. (Springer, New York), Vol.6, pp. 338-352.

3. Bowes, G., Ogren, W. L. & Hageman, R. H. (1971) Biochem.Biophys. Res. Commun. 45,716-722:

4. Lorimer, G. H. (1981) Annu. Rev. Plant Physiol. 32, 349-383.5. Laing, W. A., Ogren, W. L. & Hagemnan, R, H. (1974) PlantPhysiol,

54, 678-685.6. Somerville, C. R. & Ogren, W. L. (1979) Nature (London) 280,

833-836.7. Keys, A. J., Bird, I. F., Cornelius, M. J., Lea, P. J., Wallsgrove

R. M. & Miflin, B. J. (1978) Nature (London) 275, 741-743.8. Somerville, C. R, & Ogren, W. L. (1980) Nature (London) 286,

257-259.9. Somerville, C. R. & Ogren, W. L. (1981) Plant Physiol 67, 666-

671.10. Lehner, K. & Heldt, H. W. (1978) Biochim. Biophys. Acta 501,

531-544.11. Gimmler, H., Schafer, G., Kraminer, H. & Heber, U. (1974) Planta

120, 47-61.12. Barber, D. J. & Thurman, D. A. (1978) Plant Cell Environ. 1, 297-

303.13. Heber, U. (1974) Annu. Rev. Plant Physiol. 25, 393-421.14. Heldt, H. W. (1976) in The Intact Chloroplast, ed. Barber, J.

(Elsevier/North-Holland, Amsterdam), pp. 215-234.15. Somerville, C. R. & Ogren, W. L. (1982) in Methods in Chloro-

plast Biology, eds. Edelman, M., Hallick, R. B. & Chua, N. H.(Elsevier, Amsterdam), pp. 129-138.

16. Cossins, E. A. & Sinha, S. K. (1966) Biochem. J. 101, 542-549.17. Block, R. J., Durrum, E. L. & Zweig, G. (1955) A Manual of Pa-

per Chromatography and Paper Electrophoresis (Academic, NewYork), p. 168.

18. Spector, T. (1978) Anal. Biochem. 86, 142-146.19. Somerville, C. R., Somerville, S. C. & Ogren, W. L. (1981) Plant

Sci. Lett. 21, 89-96.20. Lilley, R. M., Fitzgerald, M. P., Rientis, K. G. & Walker, D. A.

(1975) New Phytol. 75, 1-10.21. Anderson, J. W. & Done, J. (1977) Plant Physiol 60, 354-359.22. Stokes, D. M. & Walker, D. A. (1972) Biochem. J. 128, 1147-1157.23. Heldt, H. W. (1980) Methods Enzymol 69, 604-613.24. Wintermans, J. F. G. M, & Demots, A. (1965) Biochim. Biophys.

Acta 109, 448-453.25. Fliege, R., Flugge, U. I., Werden, K. & Heldt, H. W. (1978)

Biochim. Biophys. Acta 502, 232-247.26. Somerville, C. R. & Ogren, W. L. (1980) Proc. Natl Acad. Sci. USA

77, 2684-2687.27. Somerville, C. R. & Ogren, W. L. (1982) Biochem. J. 202, 373-

380.

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